Rapid morphological change in UK populations of Impatiens glandulifera

The highly invasive Impatiens glandulifera (Himalayan balsam) is one of the most prolific and widespread invasive plants in the British Isles. Introduced in the early nineteenth century, it has now been reported in almost every vice county across the UK and is a fierce competitor that has adverse effects on the local community structure. Despite the negative impacts that invaders like I. glandulifera have on local communities, there have been very few studies which address the morphological changes that invasive plant populations have undergone since their initial introduction. This is the first study of its kind to investigate the morphological changes that have occurred in I. glandulifera. 315 herbarium specimens dating from 1865 to 2017 were used to measure changes in morphological traits such as leaf size, flower length and stomatal characteristics. We found that since 1865, there has been a significant reduction in overall leaf size, a significant reduction in stomatal density and a significant increase in the overall flower length. These results highlight the importance of monitoring the evolutionary change in prolific alien species over the course of their invasion, providing useful insights into changes in competitive ability which may prove useful in managing dispersal and providing options for potential management.

In the last 100 years, globalization and industrialisation have exponentially increased the incidence of invasions by non-native plants 1 .There is increasing concern over the resulting harm to the environment; invasive species are now regarded as a leading cause of plant extinction, second only to habitat loss [2][3][4] .Species introduced to new regions may face very different environmental conditions to those experienced in their native range, and there is evidence that many successful invasive species seem to undergo rapid phenotypic changes in response to the challenges posed by the novel/new environment 5,6 .
These rapid phenotypic changes may be the result of evolution 5 .Reproductively isolated from their source populations and faced with novel selection pressures, introduced populations must adapt quickly to their new environment to establish a stable population in their new range.Introduced populations often have a low propagule size and low genetic diversity 7,8 , which would usually reduce their evolutionary potential 9 .However, genetic bottlenecks in such invasive species can restrict gene flow 10 causing a rapid divergence of the phenotypes from their native range.Individuals that are better adapted to the new environment can appear rapidly and outcompete more poorly-adapted individuals during the lag phase of the invasion 11,12 , with adaptive traits evolving in 20 generations or less 13 .Moreover, a lack of co-evolved parasites and herbivores means non-native species may not need some costly defence strategies (e.g.secondary chemical production).This allows for resources to be invested in other traits advantageous to their new range, which will increase their competitive ability and facilitate implantation in new habitats and, consequently, their expansion into new areas [14][15][16] .
Rapid phenotypic change may also be attributed to phenotypic plasticity, resulting from genotypes producing different phenotypes in response to different environmental conditions 17 .High phenotypic plasticity is a common trait in invasive plant species, and has been demonstrated in the Himalayan balsam (Impatiens glandulifera Royle) 18 .However, regardless of whether it is caused by evolution or phenotypic plasticity, rapid phenotypic changes in response to novel environmental conditions could play a role in facilitating the spread of invasive plants 15 .Previous research that investigated phenotypic change in invasive species in response to novel www.nature.com/scientificreports/environments were based on comparisons between native and introduced populations of the same species 11,19 .Very few studies have considered the magnitude and direction of change through time since first invasion.
The present study will focus on I. glandulifera, a species native to the Himalayas that was first introduced into the UK in 1839 as a garden ornamental.It has now become a widespread and problematic invasive species found throughout most of Europe, as well as in North America, Russia, Canada and New Zealand [20][21][22][23] .I. glandulifera is a summer annual herb that reproduces only by seed and is typically found in riparian habitats in the UK.Germination takes place between February and March, with flowering occurring from July to October.The plants begin setting seed from mid-July and die back by the end of autumn with the first hard frosts 21,24 .A fierce competitor, I. glandulifera reaches up to 2.5 m tall in the UK, has a fast growth rate, over-produces nectar, and produces up to 2500 seeds per plant 21,22,25 .Forming dense monospecific stands with foliage that swamps nonnative plant species, I. glandulifera can reduce species richness in invaded habitats by 25% 26 , releasing allelopathic chemicals into the environment 27 and promoting soil erosion in riparian habitats 28 .Effective management of I. glandulifera populations in the UK requires preventing flowering for several years to deplete the seed bank.Physical (hand-pulling, mowing), chemical (herbicides) and biological methods (rust fungus, Puccinia komarovii var.glanduliferae) 29 of control are being used in the UK, with management costs estimated at approximately £1 million per annum UK-wide 23 .
In Britain, it grows taller, has higher fecundity and a larger total leaf area than in its native range, making the non-native populations more competitive than their native counterparts 30 .In this study, we used herbarium specimens to investigate changes in populations of I. glandulifera since its introduction into the U.K.This study looked at features which would give I. glandulifera an adaptive advantage, including leaf length, leaf width, leaf area, stomatal density and flower length.

Leaf area, length and width
Leaf area, leaf width and leaf length decreased with increasing time (P < 0.01; Table 1).In particular, when comparing the first four decades sampled (1860s, 1890s, 1900s, and 1910s) with the last four decades (1980s, 1990s, 2000s, and 2010s), leaf area decreased from 36.7 ± 4.3 cm 2 to 25.9 ± 1.2 cm 2 , leaf width decreased from 4.20 ± 0.24 cm to 3.48 ± 0.08 cm and leaf length decreased from 11.13 ± 0.61 cm to 9.84 ± 0.24 cm.In addition, leaf area and leaf width decreased also as a function of increasing mean temperature of the growing season (P < 0.05, Table 1, Fig. 1).However, the effects of the leaf traits in response to the mean temperature of the growing season need to be treated with caution, as the percentage of models (ran after under-sampling the last decade to N = 24) was low particularly for leaf area and leaf length.No significant effect of geographical location (as indicated by Latitude and Longitude) was found on leaf area, leaf width or leaf length (see Table 1).

Stomatal density
Stomatal density decreased significantly with increasing time (P < 0.001, Table 2), from 76 ± 4 stomata/mm 2 in the first four decades sampled (1860s, 1890s, 1900s and 1910s) to 57 ± 2 stomata/mm 2 in the last four decades decades (1980s, 1990s, 2000s and 2010s).However, this result was not supported when re-running the models after under-sampling the original dataset (Table 2, Fig. 2a).Stomatal density decreased significantly with increasing mean temperature across the growing season (P < 0.001, Table 2, Fig. 2b).No significant effect of geographical location (as indicated by Latitude and Longitude) was found on stomatal density (see Table 2).

Flower length
Flower length increased significantly with increasing time (P < 0.001, Table 2, Fig. 2c), from 2.20 ± 0.12 cm in the first four decades sampled (1860s, 1890s, 1900s, and 1910s) to 2.45 ± 0.05 cm in the last four decades (1980s,  1990s, 2000s, and 2010s).Flower length also increased with increasing mean temperature of the growing season (P = 0.006, Table 2, Fig. 2c, d).Here, again, one needs to be cautious interpreting these results, particularly with regards to changes with increasing mean temperature, as only 3% of the models re-ran after under-sampling rendered P-values < 0.05 (Table 2).No significant effect of geographical location (as indicated by Latitude and Longitude) was found on flower length (see Table 2).

Discussion
Results of the present study indicate distinct morphological changes over time in all traits measured in UK populations of I. glandulifera: leaf area, leaf length, leaf width and stomatal density have all decreased since 1865, whilst flower length increased since 1865.This suggests that there have been microevolutionary changes in UK populations of I. glandulifera, with a shift towards less competitive leaf traits, but an increase in flower length.This latter trend may reflect directional selection driven by pollinators.

Leaf length, width and area
According to the Evolution of Increased Competitive Ability hypothesis (EICA) traits such as increased height, leaf area and growth rate that give a non-native species a competitive advantage appear early during biological invasions and can result in aggressive phenotypes 31,32 .However, there have been inconsistent results in the literature on leaf characteristics in invasive species: some studies suggest that leaf area increased after the initial invasion 11,33,34 but an increasing number of studies have reported a reduction in total leaf area after invasion [35][36][37] .The results of the present study on I. glandulifera support the latter view, with leaf area, length and width all decreasing since 1865 (Fig. 1).Our study also found that leaf area and leaf width decreased with increasing mean temperature, a trend suggested by other studies 38 .A milder climate in the UK may explain the larger leaf area of I. glandulifera in the UK compared to the native range 30 .However, we might expect a decrease in leaf www.nature.com/scientificreports/area associated with higher temperatures as found in this study 39 .Smaller leaves appear to have better thermal regulation than larger leaves 40 , but regulation of leaf size is complex and can also be strongly influenced by other components of climate, such as precipitation 41 .
Although not measured in this study, a greater investment in the number of leaves produced as a result of an increase in height, could explain a reduction in leaf size as proposed by the leaf size-number trade-off theory 42 .Indeed, in the introduced range, I. glandulifera has been found to be taller than plants in the native range 30,43,44 .Several advantages have been suggested for an increase in leafing intensity, including a greater potential for higher fecundity allocation as a result of an increase in lateral inflorescences 42 .In fact, taller plants in I. glandulifera have been found to produce more seeds in UK populations 45 .In addition, taller plants may also benefit from an advantage in the competition for light 46 .

Stomatal density
Although decreases in stomatal density with time are consistent with previous findings in the literature and have been linked to the sensitivity of this trait in response to increasing CO 2 levels [47][48][49] , we must be cautious interpreting this result as it lacks support after re-running the models from datasets obtained after random re-sampling of the last decade.Decreases in stomatal density have also been reported in response to increasing temperatures, as a possible mechanism to help reduce water loss by reducing the stomatal conductance of the leaf 50 .Table 1.Estimated regression parameters, standard errors, t-values and P-values for the models performed on the leaf traits measured in I. glandulifera.P-values for the estimated regression parameters are marked in bold to denote statistical significance at P < 0.05.The adjusted R 2 , P-values for the fitted models are given in italics.Percentage of the models with P < 0.05 run on the dataset after 1000 iterations of random under-sampling to limit the last decade to n = 24.

Floral traits: flower length
Impatiens glandulifera is known for its large, self-compatible, colourful flowers that produce copious quantities of nectar with the highest sugar content of any other annual species in Europe.This has been found to negatively affect native plants by being a successful competitor for pollinators 25 .This study found that an overall significant increase in the length of I. glandulifera flowers occurred between 1865 and 2017.
Although flowers of I. glandulifera are self-compatible, they are rarely self-pollinated and have a wide range of insect pollinators, mostly bumblebees (Bombus spp.) and honeybees (Apis mellifera L.) [51][52][53] .Flowers becoming longer may result in more deeply hidden nectar, which may benefit the plant by ensuring the nectar is not stolen by non-pollinating insects and in turn increasing pollen contact with the pollinator, hence increasing the efficiency of pollination 54 .In addition, assuming high correlation among floral traits 55 , it is plausible to speculate that an increase in flower length may correlate with an increase in flower size in I. glandulifera, which might provide an adaptive advantage in its non-native range, attracting a larger number and greater diversity of pollinators,  www.nature.com/scientificreports/as has been frequently observed in other species with large flowers [56][57][58] .In fact, pollinators have been shown to have a substantial effect on directional selection of flower size across different plant families, selecting for larger, more showy flowers that produce more nectar 57,59 .Possibly bumblebees and honeybees are selectively attracted to larger flowers and visit them more frequently; research has demonstrated that larger flowers are more easily seen by bumblebees along their foraging route compared to small flowers, and the bumblebees actively visit them more frequently 56 , which may result in an increased seed set 60 .
In this study we found a positive relationship between flower length and mean temperature, although we should be cautious interpreting this result due to the lack of support when re-running the models after undersampling the original data.Elevated temperatures have been found to influence flowering traits, including pollen, nectar and flower production and also flower size 61 .However, no clear, consistent overall trends have been reported on the effects of warming on flower size, with some studies demonstrating a reduction, whilst others find an increase, pointing to species specific responses 61 .Regardless of whether the observed changes are driven by pollinators or due to increasing temperatures, it is clear that these changes may be capable of altering the interaction of I. glandulifera with pollinators.

Conclusions
This study demonstrates the importance of herbarium collections for research into the dynamics of vegetation change 62,63 .Without the collections that were investigated here, it would have been impossible to provide the temporal / historical context for the evidence obtained from fieldwork.The work has revealed how I. glandulifera has undergone rapid phenotypic changes in the UK since 1865, in particular a reduction in leaf size and an increase in mean flower length.These morphological changes can be linked to an increase in competitive ability of the species and can be partly predicted under the EICA hypothesis 32 .The reduction in leaf size may be compensated for by an increased investment in reproductive structures.In addition, the longer I. glandulifera flowers may enhance the efficiency of pollination.The next logical step would be to look at how biotic interactions have been shaping this change; are there are other biotic (and abiotic) conditions, not addressed in this study, that may also be shaping flower evolution in I. glandulifera?Future increases in mean annual temperature associated with climate change may favour these trends and promote increasing flower length.
Adaptations of I. glandulifera that promote its spread and competitive success over native species have serious ecological and financial consequences, costing the UK government around £1,000,000 annually 23 .Understanding both the ecology of an invasive species, such as I. glandulifera and, the full range of its effects on the invaded ecosystem is essential in the formulation of effective control strategies and rehabilitation of invaded habitats 64 .The main methods of management include labour-intensive pulling, cutting, herbicide treatment with glyphosate Table 2.Estimated regression parameters, standard errors, t-values and P-values for the models performed on stomatal density and flower length (cm) in I. glandulifera.P-values for the estimated regression parameters are marked in bold to denote statistical significance at P < 0.05.The adjusted R 2 , and P-values for the fitted models are given in italics., while other studies have highlighted the effectiveness of using strains of rust originating from the native range of the species 29,65 .The evolution of I. glandulifera plants with smaller leaves may influence the choice of management technique, for example, by reducing the quantity of herbicide necessary for control.The long-term consequences of the evolution of longer flowers are uncertain.I. glandulifera is favoured by beekeepers because it has an extended flowering time, coupled with high rates of sugar production, highlighting the potential use this species to support pollinating insects 66 .It has been suggested that the species has the potential to decrease genetic diversity in native plants as it lures pollinators away from natives 25 .In contrast, other research has suggested that an increase in species richness, visitor abundance and flower visitation was reported for plots invaded by I. glandulifera in multiple studies, resulting in a facilitated increase in pollinator visits for native species 64 .Further research is clearly needed to determine the impact of invasion by I. glandulifera on pollination of native species and to consider how this is affected by changes in adaptive traits highlighted by this research.

Sampling herbarium specimens
315 herbarium specimens were studied, with collection dates ranging from 1865 to 2017 (Tables S1 and S2, supplementary information).Specimens studied were located in the herbaria at Amgueddfa Cymru -Museum Wales, The Royal Botanic Garden Kew and the Natural History Museum London.Only high-quality specimens were sampled; any specimens that were badly damaged or had less than three fully intact leaves were excluded from the data analysis.Specimens were invariably from the apical portion of the plant.In this study, herbarium specimens were sampled from 41 vice-counties across the UK, covering most of Wales and southern England (Fig. 3).

Field sampling
In 2017, data was obtained from 158 field specimens collected during June-July from 11 populations across South Wales (Table S3, supplementary information).Individuals were randomly selected from each habitat and each specimen was pressed and prepared using standard herbarium methods, hence imposing similar size reduction to historical specimens 67 .The specimens were collected from public areas, predominantly parks and waste ground.Where necessary, verbal permission was obtained by contacting park rangers and wardens.Specimens collected in the field have been deposited at the herbarium (NMW) of Amgueddfa Cymru -Museum Wales, where they are available for reference by researchers and members of the public.Individual voucher numbers are as followed V.2024.002.001-V.2024.002.159.The research project complied with legislation and guidelines for research issued by Cardiff University and the U.K.

Morphological data
Digital photographs of each specimen were taken using a ruler for scale.Leaf area, length and width were all then calculated using Image J 68 .Any leaves that were located near the growing tip or that appeared immature were discounted from the analysis.Leaf length was calculated measuring from the tip of the apex to just before the petiole attachment (Fig. 4a), near the basal portion of the leaf.Leaf width was recorded as the widest portion of the leaf (Fig. 4b).The surface area of the leaf was measured as the circumference around the outside of the leaf (Fig. 4c).Where possible, five leaves were sampled per plant: where fewer leaves were available a minimum of three leaves were sampled.Flower length was calculated by measuring the flower as shown in Fig. 4d.At least three flowers were sampled per plant.
Stomatal density was measured by coating the abaxial surface of the leaf with Germolene New Skin liquid plaster, leaving to dry for five to ten minutes and gently prying off the surface with a pair of fine tipped tweezers.This peel was then examined using a Nikon Labophot-2 Binocular Phase Contrast Microscope.The full procedure for this can be found at https:// www.resea rchga te.net/ publi cation/ 32478 4278_ Non-invas ive_ method_ for_ looki ng_ at_ stoma ta_ epide rmal_ cells_ of_ herba ria_ speci mens.

Climatic data and geographic data
Mean monthly Central England Temperature (CET) records for the period 1865-2017 were obtained from the UK Meteorological Office (http:// hadobs.metoffi ce.com/ hadcet/ cetml 1659on.dat).These temperature series for Central England are representative of roughly a triangular area enclosed by Bristol, Lancashire and London 69 .It was found that monthly temperatures from stations distributed across the UK are highly correlated with the corresponding CET, indicating that its applicability extends beyond central England 70 .Longitude and latitude were calculated using the centroid of the Watsonian vice-county for each specimen.

Statistical analyses
All statistical analyses were performed in R v. 4.2.2 71 .Multiple regression models were used to explore the relationship between traits measured (leaf area, leaf width, leaf length, flower length) and time, mean temperature of the growing season (mean of the monthly averages from March to November), latitude and longitude.As time was positively correlated with mean annual temperature (Pearson's r > 0.7; Figure S1, supplementary information), different models were run for each of these two predictors to avoid issues due to multicollinearity.The package 'visreg' in R 72 was used to allow the visualization of the relationship between the response variable (partial residuals) in relation to a given predictor while holding all other variables constant.
Models were validated via diagnostic plots of model residuals to verify the assumptions of normality and homoscedasticity 73 .Log10-transformation was applied to leaf area, length and width and to flower length to meet statistical assumptions.In addition, since the experimental data was highly unbalanced due to having a high number of samples for the last decade studied, we employed a random under-sampling approach limiting

Figure 1 .
Figure 1.Partial regression plots of the effects of date of collection (Year Collected) and Mean Temperature (mean of average monthly temperatures from March to November) on Leaf Area (a and b, respectively), Leaf Width (c and d, respectively) and Leaf Length (e and f, respectively).Lines represent model predictions of statistically supported effects; grey bands indicate 95% confidence bands.The adjusted R 2 , and P-values for the fitted models are given in Table1.

Figure 2 .
Figure 2. Partial regression plots of the effects of date of collection (Year Collected) and Mean Temperature (mean of average monthly temperatures from March to November) on Stomatal Density (a and b respectively) and on Flower Length (c and d respectively).Lines represent model predictions of statistically supported effects; grey bands indicate 95% confidence bands.The adjusted R 2 , and P-values for the fitted models are given in Table2.

Figure 3 .
Figure 3.A GIS generated map showing the source of herbarium samples (i.e., vice-counties shaded in grey).

Figure 4 .
Figure 4. Morphology of I. glandulifera leaf and flower with illustrations of how each of the morphological measurements in this study were taken.(A) I. glandulifera leaf with a red line illustrating how the leaf length was calculated.(B) I. glandulifera leaf with a red line illustrating how the leaf width was calculated.(C) I. glandulifera leaf with a red line illustrating how the leaf area was calculated.(D) I. glandulifera flower with a red line illustrating how the length of the flower was calculated.